The challenge: our society is in desperate need of new approaches to combatting bacterial infections. In the first 30 years after penicillin’s discovery, >20 new classes of antibiotics were developed. This success led to the pervasive sense that we had won the war against pathogenic bacteria. But in the following years, bacteria evolved resistance to every one of these classes. Meanwhile, due to economic factors and overreliance on traditional discovery approaches, very few new antibiotics have been developed in the past 30 years. Of particular concern, no antibiotics with novel mechanisms of action (MoAs) have come to market in decades. This lack of novelty in antibiotic development has become the major problem in the field. The innovation: we have developed novel approaches to developing non-traditional antibiotics. A key bottleneck to understanding host-pathogen interactions is that infection is an asynchronous process such that bulk methods lose important information through averaging. We recently developed a breakthrough single-cell RNA-sequencing method, M3-Seq that overcomes both the scale problem and addresses the lack of polyA tails in bacterial mRNAs, enabling us to sequencing most of the mRNAs in hundreds of thousands of cells in a single experiment. We also adapted this approach to mammalian cell systems so that we can now perform dual-M3-Seq to simultaneously monitor bacterial and host gene expression in single cells. Validation: we used M3-Seq and dual-M3-Seq to understand phage dynamics and intracellular pathogenesis in vitro and in vivo. As proof of principle for our approach, we showed how M3-Seq can virtually synchronize phage infections to identify a novel anti-phage defense mechanism. We also used dual- M3-Seq to compare macrophage responses to two different intracellular pathogens, leading to the discovery of a specific sensor of Listeria monocytogenes. Proposed plans: M3-Seq as a platform for developing novel host-directed and phage therapies. Armed with the ability to monitor phage and pathogen dynamics both in vivo and in vitro at single-cell resolution, we will define why traditional phage therapies fail and understand which host pathways help combat pathogenesis in which cell types. Using this knowledge, we will then develop interventions to manipulate both phages and bacteria to improve the efficacy of phage therapy. In parallel, we will develop host-targeting cell- specific interventions that modulate the immune system to help combat infections while minimizing the emergence of resistance or complications like autoimmune reactions. This proposal has the potential to make a significant impact on both society and science, but is also risky and multidisciplinary such that it is not suited for traditional funding mechanisms. With my lab’s past track record, the Pioneer Award thus has the potential to make a powerful impact through our work.

Project Narrative: Due to rising rates of resistance to traditional antibiotics, we are in critical need of novel types of approaches for combatting life-threatening bacterial infections. Here I propose a new direction for my lab that harnesses our newfound abilities to monitor gene expression in single bacteria, mammalian cells, and phages, as well as to manipulate host gene expression in a cell-type- specific manner. We will combine these new methods to understand bacterial pathogenesis and phage therapy dynamics at unprecedented single-cell resolution and to engineer new types of nontraditional phage therapies and host-directed antibiotics to combat the rising antibiotic resistance crisis.